All Publications

Abstract

Attenuation of pre-rRNA synthesis in response to elevated temperature is accompanied by increased levels of PAPAS ("promoter and pre-rRNA antisense"), a long noncoding RNA (lncRNA) that is transcribed in an orientation antisense to pre-rRNA. Here we show that PAPAS interacts directly with DNA, forming a DNA-RNA triplex structure that tethers PAPAS to a stretch of purines within the enhancer region, thereby guiding associated CHD4/NuRD (nucleosome remodeling and deacetylation) to the rDNA promoter. Protein-RNA interaction experiments combined with RNA secondary structure mapping revealed that the N-terminal part of CHD4 interacts with an unstructured A-rich region in PAPAS. Deletion or mutation of this sequence abolishes the interaction with CHD4. Stress-dependent up-regulation of PAPAS is accompanied by dephosphorylation of CHD4 at three serine residues, which enhances the interaction of CHD4/NuRD with RNA and reinforces repression of rDNA transcription. The results emphasize the function of lncRNAs in guiding chromatin remodeling complexes to specific genomic loci and uncover a phosphorylation-dependent mechanism of CHD4/NuRD-mediated transcriptional regulation.

Abstract

Numerous studies indicate that long noncoding RNAs (lncRNAs) are dysregulated in hepatocellular carcinoma (HCC) and might serve as potential diagnostic biomarkers and therapeutic targets of HCC. Therefore, it is interesting to globally identify the lncRNAs altered in HCC. In our study, we used microarray to profile the levels of lncRNAs and mRNAs in three pairs of HCC and their adjacent noncancerous samples. We found lncRNA-SVUGP2, which is a splice variant of the UGP2 gene, was down-regulated in HCC samples and correlates with a better prognosis in patients with HCC. Overexpression of lncRNA-SVUGP2 in HepG2 and Hep3B liver cancer cells suppresses cell proliferation in vitro and tumor growth in vivo. Moreover, lncRNA-SVUGP2 suppresses the invasion ability of liver cancer cell lines and downregulates the mRNA and protein levels of MMP2 and 9. Additionally, lncRNA-SVUGP2 positively or negatively correlates with many mRNAs in liver cancer tissues, indicating it is multifunctional in regulating carcinogenesis.

Abstract

The miRNAs are small, non-coding RNAs that regulate various biological processes, including liver fibrosis. Hepatic stellate cells (HSCs) play a central role in the pathogenesis of liver fibrosis. By microarray profiling and real-time PCR, we noted that miR-31 expression in HSCs from rats, mice and humans was significantly increased during HSC activation in culture. Overall, miR-31 expression levels were unchanged in the whole-liver RNA extracts from fibrotic rat and human samples. Nevertheless, we found that miR-31 was particularly up-regulated in HSCs but not in hepatocytes during fibrogenesis. Thus, we hypothesized that miR-31 may mediate liver fibrosis. In the present study, we found that inhibition of miR-31 expression significantly inhibited HSC activation, whereas its over-expression obviously promoted HSC activation. Moreover, over-expression of miR-31 promoted HSC migration by enhancing matrix metalloproteinase (MMP)-2 expression whereas inhibition of miR-31 has an opposite effect. The biological function of miR-31 during HSC activation might be through targeting FIH1, a suppressor of hypoxia-inducible factor (HIF-1), because a knockdown of FIH1 by shRNA could mimic the effects of miR-31. In addition, primary rat HSCs were isolated and treated with different cytokines, such as transforming growth factor β (TGF-β), vascular endothelial growth factor and platelet-derived growth factor-BB, to evaluate upstream regulators of miR-31. We found that only TGF-β, a pivotal regulator in liver fibrosis, remarkably increased miR-31 expression in HSCs. And the effects of TGF-β on HSCs can be partially counteracted by inhibition of miR-31. In addition, chromatin immunoprecipitation experiments and the luciferase reporter assay demonstrated that Smad3, a major TGF-β-downstream transcription factor, stimulated the transcription activity of miR-31 by binding directly to miR-31's promoter. In conclusion, the miR-31/FIH1 pathway associates with liver fibrosis, perhaps by participation in the TGF-β/Smad3 signalling of HSCs.

Abstract

Acute myeloid leukemia (AML) is recognized as a complex disease of hematopoietic stem cell disorders, but its pathogenesis mechanisms, diagnosis, and treatment remain unclear. General histone deacetylase (HDAC) inhibitors have been used in blood cancers including AML, but the lack of gene specificity greatly limits their anti-cancer effects and clinical applications. Here, we found that HDAC1 expression was negatively correlated with that of Krüppel-like factor 4 (Klf4) and that AML patients with lower HDAC1 level had better prognosis. Further, knockdown of HDAC1 in leukemia cells K562, HL-60, and U937 significantly increased Klf4 expression and inhibited cell cycle progression and cell proliferation, similar results were found for HDAC inhibitors (VPA and mocetinostat). Moreover, overexpression or knockdown of Klf4 could markedly block the effects of HDAC1 overexpression or knockdown on leukemia cells in vitro and in vivo, respectively. Mechanistic analyses demonstrated that HDAC1 and Klf4 competitively bound to the promoter region of Klf4 and oppositely regulated Klf4 expression in myeloid leukemia. We identified HDAC1 as a potential specific target for repressing cell proliferation and inducing cell cycle arrest through interplay and modulation of Klf4 expression, suggests that HDAC1 and Klf4 are potential new molecular markers and targets for clinical diagnosis, prognosis, and treatment of myeloid leukemia.

Abstract

Growing evidence suggests that miR-29a has an important role in regulating tumourigenesis and development of various types of cancer. However, the role and the underlying mechanism of miR-29a in colorectal cancer (CRC) remain largely unknown.MiR-29a targeted gene was identified by the luciferase assay and western blot. MiR-29a function was analysed by invasion assays and the orthotopic transplantation mouse model. The miR-29a pathway was assayed by real-time PCR, western blot and chip analysis.KLF4 was identified as a direct target gene of miR-29a. MiR-29a promoted CRC cell invasion, which was blocked by re-expression of KLF4. In addition, MMP2 was identified as a novel direct target of KLF4. Both miR-29a overexpression and KLF4 knockdown promoted MMP2 expression but inhibited E-cadherin expression. Furthermore, clinical data indicated that both miR-29a high expression and KLF4 mRNA low expression were associated with metastasis and poor prognosis in CRC patients, and KLF4 protein expression was inversely correlated with MMP2 but positively correlated with E-cad protein expression.Increased expression of miR-29a promoted CRC metastasis by regulating MMP2/E-cad through direct targeting KLF4, which highlights the potential of the miR-29a inhibitor as a novel agent against CRC metastasis.

Abstract

MicroRNAs (miRNAs) have been shown to play essential roles in HSCs activation which contributes to hepatic fibrosis. Our previous miRNA microarray results suggested that miR-126 might be decreased during HSCs activation as other studies. The aim of this study is to investigate the role of miR-126 during HSCs activation.In this study, the expression of miR-126 during HSCs activation was measured and confirmed by qRT-PCR. Then, miR-126 expression was restored by transfection of lentivirus vector encoding miR-126. Futhermore, cell proliferation was assayed by the cell counting kit-8 (CCK-8), cell migration was assayed by transwell assay, and the markers of activation of HSCs, α-SMA and collagen type I, were assayed by qRT-PCR, Western Blotting, Immunostaining and ELISA. Luciferase reporter assay was used to find the target of miR-126, and Western Blotting and Immunostaining was used to validate the target of miR-126. Then, the expression and the role of the target of miR-126 during HSCs activation was further assessed.The expression of miR-126 was confirmed to be significantly decreased during HSCs activation. Overexpression of miR-126 significantly inhibited HSCs migration but did not affect HSCs proliferation. The expression of α-SMA and collagen type I were both obviously decreased by miR-126 restoration. CRK was found to be the target of miR-126 and overexpression of miR-126 significantly inhibited CRK expression. And it was found that overexpression of CRK also significantly decreased miR-126 expression and promoted HSCs activation.Our study showed that overexpression of miR-126 significantly inhibited the activation and migration of HSCs through targeting CRK which can also decrease miR-126 expression and promote HSCs activation.

Abstract

Aberrant expression of histone deacetylases (HDACs) is associated with carcinogenesis. Some HDAC inhibitors are widely considered as promising anticancer therapeutics. A major obstacle for development of HDAC inhibitors as highly safe and effective anticancer therapeutics is that our current knowledge on the contributions of different HDACs in various cancer types remains scant. Here we report that the expression level of HDAC10 was significantly lower in patients exhibiting lymph node metastasis compared with that in patients lacking lymph node metastasis in human cervical squamous cell carcinoma. Forced expression of HDAC10 in cervical cancer cells significantly inhibited cell motility and invasiveness in vitro and metastasis in vivo. Mechanistically, HDAC10 suppresses expression of matrix metalloproteinase (MMP) 2 and 9 genes, which are known to be critical for cancer cell invasion and metastasis. At the molecular level, HDAC10 binds to MMP2 and -9 promoter regions, reduces the histone acetylation level, and inhibits the binding of RNA polymerase II to these regions. Furthermore, an HDAC10 mutant lacking histone deacetylase activity failed to mimic the functions of full-length protein. These results identify a critical role of HDAC10 in suppression of cervical cancer metastasis, underscoring the importance of developing isoform-specific HDAC inhibitors for treatment of certain cancer types such as cervical squamous cell carcinoma.

Abstract

Aberrant expression of mitotic checkpoint genes compromises mitotic checkpoint, leads to chromosome instability and tumorigenesis. However, the cell signals that control mitotic checkpoint gene expression have not been reported so far. In the present study we show that, in human breast cancer cells, chemical inhibition of Bone morphogenetic proteins (BMPs), but not Transforming Growth Factor-β (TGF-β), abrogates the mitotic arrest induced by nocodazole. Protein expression analysis reveals that inhibition of BMP signaling dramatically down regulates protein levels of mitotic checkpoint components BUB3, Hec1, TTK and MAD2, but inhibition of TGF-β has relatively minor effect on the expression of these proteins. Activation of BMP signaling specifically up regulates BUB3, and activation of Activin A signaling globally down regulates these proteins level. Furthermore, overexpressing MAD2, TTK, BUB3 or Hec1 significantly rescues the mitotic arrest defect caused by BMP inhibition. Our results demonstrated for the first time that TGF-β family cytokines are cellular signals regulating mitotic checkpoint and perturbations in intrinsic BMP signaling could lead to suppression of mitotic checkpoint signaling by downregulating key checkpoint proteins. The results suggest a possible mechanism by which dysregulation of TGF-β signaling causes mitotic checkpoint defects and drives tumorigenesis. The finding also provides a potential and more specific strategy for cancer prevention by targeting BMP and mitotic checkpoint connection.

Abstract

Cell migration plays major roles in human breast cancer-related death, but the molecular mechanisms remain unclear. Valproic acid (VPA) is a broad-spectrum inhibitor of class I and II histone deacetylases and shows great anticancer activity in a variety of human cancers including breast cancer. In this study, we found that VPA significantly inhibited cell migration but not proliferation of human breast cancer MDA-MB-231 cells. Mechanistic studies found that VPA significantly inhibited the expression of Survivin. Knockdown of Survivin could obviously inhibited cell migration, while over-expression of Survivin markedly rescued the inhibition of VPA on cell migration. Further studies found that knockdown of HDAC2 completely mimicked the effects of VPA on Survivin and cell migration, and over-expression of Survivin could also rescue the effects of HDAC2 knockdown on cell migration. Collectively, these results indicated that HDAC2 may be the specific target of VPA in breast cancer cells, and specific inhibition of HDAC2, especially by small molecular chemicals may lead to less side-effects and provide a better strategy than VPA application for human breast cancer treatment.

Abstract

E-Cadherin plays important roles in cell-cell adhesion, epithelial-to-mesenchymal transition, cancer cell migration and invasion. Valproic acid (VPA), a well-known inhibitor of class I and class II histone deacetylases, has been considered a promising anticancer drug due to its capacity of inducing cancer cell proliferation arrest and death through different mechanisms. However, effects of VPA on E-cadherin mediated cell-cell adhesion and cancer cell migration remain unclear. In the present study, we found that VPA potently induced hyperacetylation of histone H3 and H4, increased the expression of E-cadherin and inhibited cell migration in prostate cancer cells. Furthermore, knock-down of E-cadherin significantly restored the effects of VPA on cell migration, while over-expression of E-cadherin in prostate cancer cells significantly inhibited cell migration to a similar level as VPA treatment. These results thus suggest that up-regulation of E-cadherin and inhibition of cell migration may represent a new anticancer mechanism of VPA.